Perturbation in the replication-stress response (RSR) and DNA damage response (DDR) causes genomic-instability. Replication protein A (RPA) is a single-strand DNA (ssDNA) binding protein with key roles in the RSR and DDR. Genomic-instability occurs in Wiskott-Aldrich syndrome (WAS), a primary immunodeficiency and cancer susceptibility disorder, yet the molecular underpinnings of unstable genome in WAS cells remain uncharacterized. WASp, the protein deficient in this disorder, functions both in the cytoplasm and nucleus. In the nucleus, WASp functions to prevent the accumulation of harmful R loops (RNA-DNA hybrids + ssDNA) and in the pre-repair step of escorting broken DNA ends to the repair sites by the homology-directed repair (HDR) pathway. Accordingly, WAS patient lymphocytes are poorly-equipped to both prevent and resolve DNA damage, which proposes WAS as a “genotoxin-sensitive” immune dysregulation disorder. Our foundational studies have uncovered an essential role of WASp in the RSR by influencing RPA functions under hydroxyurea-induced replication stress, in both immune and nonimmune cells. Therefore, the overall objective of this proposal is to explicate the molecular details of how WASp safeguards normal replication and which proteins and pathways WASp associates with to enable this function during replicative stress. We will test the hypothesis that WASp is required to both prevent and manage replication stress and DNA damage. Specifically, we will define WASp role in mechanisms that process a blocked replication fork (in Aim 1) and address how WASp role specifically on modifying the actin state (G-actin vs. F-actin) influence RSR (in Aim 2). Achieving these aims will propose WASp as a novel RSR factor, and human WAS as a disease of dysfunctional RSR, which may provide new mechanisms for oncogenesis in WAS.